Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins

Figures

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  Fig. 1. Cryo-EM structure of the inverted acidic domain of cortactin bound to Arp2/3 complex. a Domain diagram of human cortactin (UniProt: Q60598), highlighting the sequence of the fragment Cort1-76 used in the cryo-EM structure and residues D20-V29 implicated in Arp2/3 complex binding. b Cryo-EM map of Cort1-76 (orange) bound to Arp2/3 complex. Arp2/3 complex subunits are colored as follows: Arp2, cyan; Arp3, green; ArpC1, yellow; ArpC2, ArpC3, and ArpC5, light gray; ArpC4, dark gray. The inset shows cortactin residues D20-V29 and the corresponding cryo-EM map in orange, and the binding site on Arp3 (green cartoon representation with gray side chains). See also Supplementary Fig. 2a. c Superimposition of the A domains of cortactin (orange), N-WASP (magenta, PDB code 6UHC), and Arpin (blue, PDB code 7JPN) on Arp3 (green and gray). Insets show the binding site (gray) of the A domains and the binding pocket of the conserved tryptophan of the three proteins. The sequences of the three A domains are shown on the side, vertically aligned according to the structures. Note that cortactin binds the site on Arp3 with inverted polarity compared to N-WASP and Arpin.

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  Fig. 2. Effect of Cort1-76 and N-WASP WCA on the short-pitch transition of Arp2/3 complex. Western blot analysis (Arp3 antibody, left) and densitometric quantification (n = 3, right) of the cross-linked fraction of Arp2/3 complex alone (black) or with WCA (magenta), Cort1-76 (orange), or WCA and Cort1-76 (green). All the Western blots used for quantification are shown in Supplementary Fig. 3. Data were fit to a first-order exponential equation (kinetic parameters of the fit listed in the figure) and are reported as mean value ± standard deviation. The statistical significance of the measurements was calculated using an unpaired, two-tailed t-test (P-values listed in the figure). For each data point, P-values for pairwise comparisons with Arp2/3 complex alone (control) are shown from top-to-bottom for experiments with WCA + Cort1-76, WCA, or Cort1-76 (ns, non-significant). The source data are provided as a Source Data file.

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  Fig. 3. Arp2/3 complex activation by cortactin constructs with or without monomeric N-WASP WCA. a Densitometric quantification of cosedimentation experiments (left) of Arp2/3 complex with F-actin in the absence (black) or the presence of the cortactin constructs schematically shown on the right (color-coded). A characterization of the cortactin constructs by SDS-PAGE and the cosedimentation experiments used for quantification are shown in Supplementary Figs. 4, 7, respectively. The curves represent fits to a first-order exponential equation. For each data point, P-values for pairwise comparisons with Arp2/3 complex alone (control) are shown from top-to-bottom for experiments with Cort1-401, Cort1-79/154-401, Cort1-227, or Cort1-76 (ns, non-significant). b–e Left, Time-course of actin polymerization under the following conditions: actin alone (black), actin + Arp2/3 complex (light gray), actin + Arp2/3 complex + indicated cortactin constructs (dark gray), actin + Arp2/3 complex + monomeric N-WASP WCA (magenta), actin + Arp2/3 complex + WCA + indicated cortactin constructs at increasing concentrations (color coded as in part a, with darker shades representing higher concentrations as…

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  Fig. 4. Arp2/3 complex activation by N-terminal cortactin constructs with or without dimeric N-WASP WCA. a–c Top, Time-course of actin polymerization under the following conditions: actin alone (black), actin + Arp2/3 complex (light gray), actin + Arp2/3 complex + indicated cortactin constructs (dark gray), actin + Arp2/3 complex + GST-WWCA (magenta), actin + Arp2/3 complex + GST-WWCA + indicated cortactin constructs at increasing concentrations (darker shades represent higher concentrations as indicated). Data are shown as the average curve from three independent experiments with s.d. error bars in lighter color. Bottom, Maximum polymerization rates calculated from the polymerization curves shown on the top (n = 3). Source Data are provided as a Source Data file. The statistical significance of the measurements was calculated using an unpaired, two-tailed t-test (P-values listed in the figure; ns, non-significant). d ITC titrations of cortactin constructs Cort1-76 (left), Cort18-76 (middle), and Cort1-33 (right) into ATP-Arp2/3 complex. The experimental conditions, including temperature (T), protein concentration, and fitting parameters (stoichiometry, N and dissociation constant, KD)…

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  Fig. 5. Mechanism of cortactin coactivation of Arp2/3 complex with NPFs and branch stabilization. Actin-loaded monomeric (top) or dimeric (bottom) NPFs bind inactive Arp2/3 complex through the high-affinity site 1 on Arp2-ArpC1. Cortactin competes with NPF binding to site 2 on Arp3, but not to site 1. NPF binding to Arp2-ArpC1 shifts the equilibrium of Arp2/3 complex toward the active, short-pitch conformation and delivers actin at the barbed end of Arp2, whereas cortactin recruits this “primed” complex to the mother filament. In the case of monomeric NPFs, the binding of actin at the barbed end of Arp3 is diffusion limited. For dimeric NPFs, however, one arm of the dimer is bound to Arp2-ArpC1, whereas the other arm is close enough to deliver actin to Arp3. Binding of the primed complex to the side of the mother filament shifts the equilibrium further in the direction of the active, short-pitch conformation. The actin subunits that begin adding at the barbed end of the Arps compete with NPF binding to sites 1 and 2, causing NPF release. Upon NPF release, the short-pitch conformation is fully stabilized through interactions of Arp2/3 complex with actin subunits of the mother and daughter…